We present the nELISA, a high-throughput, high-fidelity, and high-plex protein profiling platform. DNA oligonucleotides are used to pre-assemble antibody pairs on spectrally encoded microparticles and perform displacement-mediated detection. Spatial separation between non-cognate antibodies prevents the rise of reagent-driven cross-reactivity, while read-out is performed cost-efficiently and at high-throughput using flow cytometry. We assembled an inflammatory panel of 191 targets that were multiplexed without cross-reactivity or impact on performance vs 1-plex signals, with sensitivities as low as 0.1pg/mL and measurements spanning 7 orders of magnitude. We then performed a large-scale secretome perturbation screen of peripheral blood mononuclear cells (PBMCs), with cytokines as both perturbagens and read-outs, measuring 7,392 samples and generating ~1.5M protein datapoints in under a week, a significant advance in throughput compared to other highly multiplexed immunoassays. We uncovered 447 significant cytokine responses, including multiple putatively novel ones, that were conserved across donors and stimulation conditions. We also validated the nELISA's use in phenotypic screening, and propose its application to drug discovery.